Integrase (IN) is encoded by the Pol gene from the HIV provirus and can be efficiently expressed as an active recombinant protein. Our laboratory has pioneered the integrase inhibitors research field (PNAS 1993), discovered several families of lead inhibitors (Nature Rev Drug Discovery 2005; Current Topics in Medicinal Chemistry 2009; Viruses 2010; Adv Pharmacol 2013), demonstrated that IN inhibitors act as interfacial inhibitors (Nature Rev Drug Discovery 2012), and patented compounds for therapeutic development. Our current studies are focused on the discovery of novel chemotype integrase inhibitors to overcome resistance to raltegravir and target novel sites of IN. We have discovered novel chemotypes derived from short Vpr peptides, which act as 3'-processing and strand transfer inhibitors. We have shown they could serve as antivirals by adding a poly-arginine tail to confer cellular uptake. A long-term goal is to build non-peptidic derivatives of those Vpr peptides. We have also published and patented novel synthetic chemotypes as IN strand transfer inhibitors (INSTIs) including phtalimide and quinolinonyl derivatives in collaborations with Dr. Terrence Burke, Laboratory of Medicinal Chemistry (CCR, NCI). To perform these experiments, we have developed a panel of recombinant IN proteins bearing the mutations observed in patients that develop resistance to raltegravir and elvitegravir. Using our set of raltegravir- and elvitegravir-resistant IN mutants, we have characterized the molecular pharmacology of elvitegravir, dolutegravir and our novel inhibitors, comparing them to raltegravir. We have shown that raltegravir, elvitegravir, dolutegravir and our novel series are highly selective for the strand transfer reaction, while being more than 100-fold less potent against the 3'-processing reaction, and almost inactive against the disintegration reaction mediated by integrase. The selective activity against strand transfer (one of the 3 reactions mediated by integrase) demonstrates the very high specificity of the clinically developed strand transfer inhibitors. It is consistent with our pharmacological hypothesis (Nature Drug Discovery 2012) that the strand transfer inhibitors trap the IN-viral DNA complex by chelating the divalent metals in the enzyme catalytic site following 3'-processing of the viral DNA and with our co-crystal structure and molecular modeling data. We have characterized the biochemical enzymatic activities and drug sensitivities of the IN mutants that confer clinical drug resistance. We have expanded these studies to double-mutants in the integrase flexible loop that commonly arise in raltegravir-resistant patients. The working hypothesis is that the second mutation acts as gain of function to rescue the biochemical activity of IN after it had become defective by the presence of the first mutation. One of aims is to understand the molecular mechanisms of such complementation and the structural connections between the flexible loop, the viral and host DNAs, and the inhibitors. We found that the flexible loop double-mutant 140S-148H is cross-resistant to both raltegravir and elvitegravir but much less to dolutegravir and to some of our new derivatives. On the other hand, the 143Y mutant is primarily resistant to raltegravir and minimally resistant to elvitegravir and dolutegravir. These results provide a rationale for using elvitegravir in patients that develop resistance to raltegravir due to mutation 143Y (but not in the case of mutations 140S-148H). Our results support the value of dolutegravir to overcome resistance to raltegravir and elvitegravir and facilitate patient compliance. To elucidate the structural basis for the potency and rational design of IN inhibitors, we determined crystal structures of wild type and mutant prototype foamy virus intasomes bound to the drugs. This work was done in collaboration with Dr. Peter Cherepanov at the Clare Hall Cancer UK Center in London. The abilityto structurally adapt to the structural changes associated with drug resistance appears to be a desirable characteristic that could be used in the development of our new INSTIs. Our studies are the result of our long-term collaboration with Dr. Terrence Burke (Chemical Biology Laboratory, CCR-NCI), with Dr. Stephen Hughes, also at the NCI-Frederick Laboratory (HIV Drug Resistance Program), and with Dr. Peter Cherepanov in London.